文章摘要
王 欢,刘琼琼,唐小军,徐 鑫,褚 楚,熊四平,郑 峰,童 华,朱 进,冯振卿,林 红.人源抗TROP2全分子抗体IgG的真核表达及对胰腺癌细胞增殖的抑制作用[J].南京医科大学学报,2014,(7):863-869~882
人源抗TROP2全分子抗体IgG的真核表达及对胰腺癌细胞增殖的抑制作用
Eukaryotic expression of human anti-TROP2 antibody IgG and its inhibitory effect on cell proliferation of pancreatic cancer
投稿时间:2014-03-17  
DOI:10.7655/NYDXBNS20140703
中文关键词: TROP2  真核表达系统  CHO dhfr-细胞  胰腺癌
英文关键词: TROP2  eukaryotic expression system  CHO dhfr- cell line  pancreatic cancer
基金项目:国家自然科学基金项目(81101704);南京市医学科技发展项目(ZKX12025)
作者单位
王 欢 江苏大学基础医学与医学技术学院,江苏 镇江 212013 
刘琼琼 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
唐小军 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
徐 鑫 江苏大学基础医学与医学技术学院,江苏 镇江 212013 
褚 楚 南京医科大学附属南京妇幼保健院妇科,江苏 南京 210004 
熊四平 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
郑 峰 南京军区军事医学研究所,江苏 南京 210002 
童 华 南京医科大学附属南京妇幼保健院妇科,江苏 南京 210004 
朱 进 南京军区军事医学研究所,江苏 南京 210002 
冯振卿 江苏大学基础医学与医学技术学院,江苏 镇江 212013
南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
林 红 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
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中文摘要:
      目的:以人源抗TROP2抗体Fab基因为模板,构建人源抗TROP2全分子抗体IgG真核表达系统,观察人源抗TROP2全分子抗体IgG对胰腺癌细胞增殖的抑制作用?方法:分别扩增抗TROP2抗体的重链和轻链基因,构建人源抗TROP2全分子抗体IgG的重组表达载体pWS-anti-TROP2,转染CHO dhfr-细胞,加MTX筛选抗体表达量高的单克隆株,Protein G 亲和柱纯化,获得人源抗TROP2全分子抗体IgG?SDS-PAGE?Western blot?ELISA?免疫荧光和流式细胞术等方法鉴定人源抗TROP2全分子抗体IgG并分析其免疫学活性?MTT法分析人源抗TROP2全分子抗体IgG对胰腺癌细胞BxPC-3的增殖抑制作用?结果:成功构建了人源抗TROP2全分子抗体IgG的真核表达系统,表达并纯化人源抗TROP2全分子抗体IgG?经鉴定,抗体的轻链与重链大小与预期一致,可与TROP2蛋白特异性结合,效价可达1∶6 400?MTT检测结果表明,人源抗TROP2全分子抗体IgG对胰腺癌BxPC3细胞的增殖有明显的抑制作用,且呈时效?量效依赖关系?结论:本研究成功构建了人源抗TROP2全分子抗体IgG的真核表达系统,并证明此抗体可特异性识别胰腺癌细胞表面的TROP2蛋白,且对胰腺癌细胞的增殖有明显的抑制作用?
英文摘要:
      Objective:To construct a eukaryotic expression system of human anti-TROP2 antibody IgG by using human anti-TROP2 Fab antibody gene as template,so as to study its inhibition of pancreatic cancer cell proliferation. Methods:The recombinant expression vector of human anti-TROP2 antibody IgG,named as pWS-anti-TROP2,was established by amplifying the heavy chain and light chain genes of human anti-TROP2 antibody IgG,respectively. pWS-anti-TROP2 plasmids were transfected into CHO dhfr- cell lines,and the monoclonal cell strains with high antibody expression were harvested by MTX screening. Then,human anti-TROP2 antibody IgG was obtained using Protein G affinity purification. The identification and immunological characteristics of the human anti-TROP2 antibody IgG were analyzed by several methods,including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),Westent blotting assay,enzyme-linked immuno sorbent assay(ELISA),flow cytometry method(FCM) and immuno fluorescence assay. The inhibition function of human anti-TROP2 antibody IgG in proliferation of BxPC3 cells was analyzed by MTT assay. Results:The eukaryotic expression system of human anti-TROP2 antibody IgG was constructed successfully. Purified human anti-TROP2 antibody IgG was obtained. The molecular weight of light and heavy chains of human anti-TROP2 antibody IgG are consistent with expected results,the antibodies could bind to TROP2 protein specifically,and the antibody titer reached 1∶6 400. MTT assay demonstrated that human anti-TROP2 antibody IgG played a significant pole in inhibiting BxPC3 cell proliferation,and the inhibition ratio was gradually increased with prolonged time and increased antibody dose. Conclusion:In the study,the eukaryotic expression system of human anti-TROP2 antibody IgG was established successfully,and it was proved that the antibody could recognize native TROP2 protein on pancreatic cancer cell surface specificially and play an important role in inhibiting the proliferation of pancreatic cancer cells.
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